Cells transformed or transfected with HCMV US2 gene

ABSTRACT

Infection of human fibroblast cells with human cytomegalovirus (HCMV) causes down-regulation of cell surface expression of MHC class I. A recombinant mutant HCMV which fails to down-regulate class I heavy chain expression is described. A method of controlling down-regulation of MHC class I expression in a cytomegalovirus infected cell, a pharmaceutical composition, a vaccine composition, 
     a method of preventing or reducing susceptibility to acute cytomegalovirus in an individual, and a virus based gene therapy vector are also described.

This is a divisional of application Ser. No. 08/509,214 filed on Jul.31, 1995, now U.S. Pat. No. 5,843,458, which is a continuation-in-partof application Ser. No. 08/282,696, filed on Jul. 29, 1994, the entiredisclosures of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to recombinant mutant humancytomegalovirus (HCMV) which does not down-regulate expression ofcellular MHC class I heavy chains upon infection and the identificationof two human cytonegalovirus gene products sufficient to cause suchdown-regulation.

BACKGROUND OF THE INVENTION

Human cytomegalovirus (HCMV) is a betaherpesvirus which causesclinically serious disease in immunocompromised and immunosuppressedadults, as well as in some infants infected in utero or perinatally(Alford and Britt, 1990). In human cytomegalovirus (HCMV)-infectedcells, expression of the cellular major histocompatibility complex (MHC)class I heavy chains is down-regulated. The 230-kb dsDNA genome of HCMVwas sequenced (Chee et al., 1990) and has at least 200 open readingframes (ORFs). The functions of most of these 200 genes is unknown. Forpurposes of this application, open reading frame is defined as theportion of a gene which encodes a string of amino acids and hence mayencode a protein. The function of some HCMV proteins are known orpredicted due to their homology with other viral (esp. herpes simplexvirus) and cellular proteins. However, for the majority of the HCMVORFs, the function(s) of the proteins they encode is unknown.

In order to study HCMV gene function, HCMV deletion mutants can beconstructed in order to assess their in vitro growth properties (Joneset al., 1991; Jones and Muzithras, 1992). For purposes of thisapplication, deletion mutants are defined as human cytomegalovirusmutants which lack regions of the wild-type viral genome. This strategyinvolves site-directed replacement mutagenesis of selected HCMV gene(s)by a prokaryotic reporter gene, usually, β-glucuronidase, althoughguanosine phosphoribosyltransferase can also be used. In this fashion,the recombinant virus can be isolated only if the replaced viral gene(s)is nonessential.

Several investigators have shown that infection by HCMV results in thedown-regulation of cellular MHC class I heavy chains (Browne et al.,1990; Beersma et al., 1993; Yamashita et al., 1993). For purposes ofthis application, down-regulation is defined as reduction in eithersynthesis, stability or surface expression of MHC class I heavy chains.Such a phenomenon has been reported for some other DNA viruses,including adenovirus, murine cytomegalovirus, and herpes simplex virus(Anderson et al., 1985; Burget and Kvist, 1985; del Val et al., 1989;Campbell et al., 1992; Campbell and Slater, 1994; York et al., 1994). Inthe adenovirus and herpes simplex virus systems, the product of a viralgene which is dispensable for replication in vitro is sufficient tocause down-regulation of MHC class I heavy chains (Anderson et al.,1985; Burget and Kvist, 1985). The gene(s) involved in class I heavychain down-regulation by murine cytomegalovirus have not yet beenidentified.

SUMMARY OF THE INVENTION

The present invention provides a recombinant mutant humancytomegalovirus which does not down-regulate expression of cellular MHCclass I heavy chains upon infection. A region of the genome of therecombinant cytomegalovirus (HCMV) mutant containing open reading frameUS2 has been deleted.

The present invention also provides a method of controllingdown-regulation of major histocompatibility complex (MHC) class Iexpression in a cytomegalovirus infected cell which utilizes therecombinant mutant human cytomegalovirus.

The present invention also provides a vaccine which utilizes therecombinant mutant human cytomegalovirus, as well as a method ofimmunizing an individual against cytomegalovirus employing therecombinant mutant human cytomegalovirus. A live attenuated HCMV vaccinelacking this gene region of open reading frame US2 will elicit a betterimmune response than one containing this gene region, based on the lackof class I down-regulation by the former. Therefore a virus lacking thisregion is a superior immunogen.

The present invention also provides a method of preventing or reducingsusceptibility to acute cytomegalovirus in an individual byadministering an immunogenic amount of the recombinant mutant humancytomegalovirus.

The present invention also provides a gene therapy vector in which theopen reading frame US2 of the HCMV gene involved in the MHC class Iheavy chain down-regulation can be incorporated into adenovirus vectorsor similar virus based gene therapy vectors to minimize the immuneresponse. This will allow the use of the recombinant adenovirus orsimilar virus based gene therapy vectors to be used in gene therapy.

The invention may be more fully understood by reference to the followingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1J show organization of recombinant virus genomes. FIG. 1A, thefirst line, is a schematic of the overall organization of the HCMVwild-type genome. Unique region sequences are shown by a line, whilerepeated region sequences are indicated by shaded boxes. RelevantHindIII fragments, within the L and S components, are indicated byletter designation (Oram et al., 1982). The second line is an expansionof the wild-type HindIII-Q, -X, and -V regions of the S component. Thesignificant open reading frames, and their orientation, are shown asopen boxes (Chee et al., 1990). The position of the IRs repeatedsequences is indicated by the shaded rectangle. The locations of Hindlel(H) and XhoI (X) restriction endonuclease sites are shown. FIGS. 1B-Ishow the genomic organization of the indicated HCMV mutant. In eachcase, the first line is the organization of the parental AD169 wild-typegenome, the second line represents the organization of relevantsequences of the linearized plasmid used to make the recombinant virus.The slanted lines indicate the boundaries of the viral flankingsequences which may be involved in homologous recombination to createthe desired mutation. The region deleted is indicated by a shaded boxbelow the first line. FIG. 1J shows the derivation and organization ofRV799. The first two lines are the same representations as FIGS. 1B-I,with the third line representing the organization of the relevantsequences of the linearized plasmid used to make RV799 from the RV134parent (second line). Restriction sites are: ApaI (A), AatII (Aa), BsmI(Bs), HindIII (H), HpaI (Hp), NarI (Na), NcoI (Nc), NheI (Nh), PstI (P),SaII (S), SauI (Sa), SphtI (Sp), SstI (T1), SstII (T2), XbaI (Xb), andXhoI (X).

FIG. 2A-2C show organization of recombinant virus genomes. FIG. 2A, thefirst line, is a schematic of the overall organization of the HCMVwild-type genome. Unique region sequences are shown by a line, whilerepeated region sequences are indicated by shaded boxes. RelevantHindill fragments, within the L and S components, are indicated byletter designation (Oram et al., 1982). The second line is an expansionof the wild-type HindIII-Q, -X, and -V regions of the S component. Thesignificant open reading frames, and their orientation, are shown asopen boxes (Chee et al., 1990). The position of the IR_(s) repeatedsequences is indicated by the shaded rectangle. The locations of HindIII(H) and XhoI (X) restriction endonuclease sites are shown. FIGS. 2B and2C show the genomic organization of the indicated HCMV mutant. In eachcase, the first line is the organization of the parental AD169 wild-typegenome, the second line represents the organization of relevantsequences of the linearized plasmid used to make the recombinant virus.The slanted lines indicate the boundaries of the viral flankingsequences which may be involved in homologous recombination to createthe desired mutation. The region deleted is indicated by a shaded boxbelow the first line. Restriction sites are: EcoRV (RV), HindIII (H),KpnI (K), PstI (P), Sacl (Sa), SmaI (Sm), XbaI (Xb), and XhoI (X). Sitesin parentheses no longer exist in the recombinant virus.

FIG. 3A-3E show the detection of cell surface MHC class I byimmunofluorescence-flow cytometry in HCMV-infected cells. Human foreskinfibroblast (HFF) cells were infected with the indicated virus at amultiplicity of infection of 5 PFU/cell for 72 h. At that time, cellswere fixed in 1% paraformaldehyde and stained with primary antibodyspecific for HLA-A, -B, -C (W6/32) or control mouse IgG (isotypematched) followed by secondary FITC-conjugated goat anti-mouse IgG.Percent positive cells (5×10³ total) and mean fluorescent intensity(MFI) were calculated on the basis of forward angle light scatter versuslog-integrated 90° light scatter using the Immuno Program, Coulter MDADSI.

FIG. 4A-4C show expression of MHC class I heavy chains in HCMV wild-typestrain AD169-infected cells. FIG. 4A is a Western blot analysis. HFFcells were uninfected (U) or infected at a multiplicity of infection of5 PFU/cell. At 24, 48, and 72 h post-infection, total cellular proteinswere harvested, electrophoresed through a 15% SDS-polyacrylamide gel,electroblotted to nitrocellulose, and probed with TP25.99 murinemonoclonal antibody (specific for a non-conformational epitope on MHCclass I heavy chains) using an ECL chemiluminescent detection kit(Amersham). FIGS. 4B and 4C are immunoprecipitation analyses. HFF cellswere uninfected or infected (as above), either in the absence orpresence (+PFA) of phosphonoformate and radiolabeled either for 4 h atlate times post-infection (69-73 h) (FIG. 4B) or for 2 h at theindicated time post-infection (FIG. 4C). Proteins were harvestedimmediately after radiolabeling and class I heavy chains wereimmunoprecipitated using TP25.99 murine monoclonal antibody.

FIG. 5A-5C shows the analysis of heavy chain expression in cellsinfected with HCMV mutants. HFF cells were uninfected (U) or infectedwith the indicated virus (multiplicity of infection of 5 PFU/cell) andradiolabeled for 4 h at late times post-infection (69-73 h). Proteinswere harvested immediately after radiolabeling. FIG. 5A is a radiographof class I heavy chains which were immunoprecipitated using TP25.99murine monoclonal antibody. FIG. 5B is a radiograph of totalradiolabeled proteins to verify approximately equivalent radiolabelingefficiency. FIG. 5C is a radiograph to verify equal progression throughthe viral replicative cycle. UL80 proteins were immunoprecipitated usinganti-assembly protein rabbit polyclonal antiserum.

FIG. 6A-6C show immunoprecipitation of class I heavy chains from RV798-,RV799-, RV134-, or AD169 wild-type-infected cells. HFF cells wereuninfected (U) or infected with the indicated virus (multiplicity ofinfection of 5 PFU/cell) and radiolabeled for 2 h at late timespost-infection (71-73 h). Proteins were harvested immediately afterradiolabeling. FIG. 6A is a radiograph of class I heavy chains whichwere immunoprecipitated using TP25.99 murine monoclonal antibody.Equivalent radiolabeling efficiency (FIG. 6B) and progression throughthe viral replicative cycle (FIG. 6C) were verified as described forFIG. 5B and C.

FIG. 7 is a radiograph showing the endoglycosidase H sensitivity ofclass I heavy chains synthesized in RV798-infected cells. HFF cells wereinfected with RV798 (multiplicity of infection of 5 PFU/cell) andradiolabeled for 2 h at early times (6-8 h) or late times (80-82 h)post-infection. For comparison purposes, uninfected cells wereradiolabeled for 2 h. Proteins were harvested either immediately afterradiolabeling (pulse) or after a 2 h chase (chase) in complete unlabeledmedia. Class I heavy chains were immunoprecipitated using TP25.99 murinemonoclonal antibody. Immunoprecipitated protein were incubated for 6 heither in the presence (+) or absence (-) of 1.5 mU of endoglycosidaseH, prior to SDS-polyacrylamide gel electrophoresis and fluorography.

FIG. 8A-8C show the immunoprecipitation of class I heavy chains fromRV798-, RV7181-, RV7177-, or AD169 wild-type-infected cells. HFF cellswere uninfected (U) or infected with the indicated virus (multiplicityof infection of 5 PFU/cell) and radiolabeled for 2 h at late timespost-infection (65-67 h). Proteins were harvested immediately afterradiolabeling. FIG. 8A is a radiograph of class I heavy chains whichwere immunoprecipitated using TP25.99 murine monoclonal antibody.Equivalent radiolabeling efficiency (FIG. 8B) and progression throughthe viral replicative cycle (FIG. 8C) were verified as described forFIG. 5B-C.

FIG. 9 provides a summary of MHC class I heavy chain expression datafrom HFF cells infected with wild-type and mutant HCMV. In FIG. 9A, thefirst line is the overall organization of the HCMV wild-type genome, andthe second line is an expansion of the wild-type HindIII-Q and -Xregions of the S component. The ORFs are indicated by an unshadedrectangle; the unlabeled ORF overlapping US4 and US5 is US4.5. In FIG.9B, the deletions within the various HCMV mutants are indicated by theshaded rectangle. RV670 is deleted of IRS1-US9 and US11; RV35 is deletedof US6-US11; RV67 is deleted of US10-US11; RV80 is deleted of US8-US9;RV725 is deleted of US7; RV69 is deleted of US6; RV47 is deleted ofUS2-US3; RV5122 is deleted of US1; RV46 is deleted of IRS1; RV798 isdeleted of US2-US11; RV7181 is deleted of IRS1-US9; RV7177 is deleted ofIRS1-US6; and RV7186 is deleted of IRS1-US11. MHC class I heavy chaindown-regulation results are from immunoprecipitation experiments (usingthe heavy chain conformation-independent monoclonal antibody, TP25.99)in which HCMV-infected HFF cells were radiolabeled at late timespost-infection. FIG. 9C shows the location of the two subregions whichcontain gene(s) which are sufficient for MHC class I heavy chaindown-regulation. Subregion A contains ORFs US2-US5 (bases 193119-195607)and subregion B contains ORFs US10 and US11 (bases 199083-200360).

FIG. 10A-10D are photographs which show localization of US11 geneproduct (gpUS11) in infected cells by immunofluorescence. HFF cells wereuninfected or infected with either AD169 wild-type or RV699 (deleted ofthe US11 gene) at a multiplicity of infection of 5 PFU/cell. After 8 h,uninfected and infected cells were fixed with 4% paraformaldehyde. Somecells were then permeabilized with 0.2% Triton X-100. The primaryantibody was rabbit polyclonal antisera raised against a US11 fusionprotein (Jones and Muzithras, 1991). Fluorescence was visualized througha Zeiss microscope.

FIG. 11A-11C show immunoprecipitation of MHC class I heavy chains fromcells infected with HCMV wild-type and mutants. HFF or U373-MG cellswere uninfected (U) or infected with the indicated virus (multiplicityof infection of 5 or 3 PFU/cell, respectively) and radiolabeled for 4 hat the indicated time post-infection. Infected cell proteins wereextracted immediately after radiolabeling and subject toimmunoprecipitation by the anti-human MHC class I heavy chain monoclonalantibody TP25.99. HFF cells were radiolabeled at either early times(FIG. 11A) or late times (FIG. 11B) post-infection. U373-MG cells wereradiolabeled at late times post-infection (FIG. 11C). AD169 is the HCMVwild-type strain from which the deletion mutant viruses were derived.RV699 is deleted of the US11 gene only; RV35 is deleted of US6 throughUS11 genes; RV8146 is deleted of US4 through US11; RV8173 is deleted ofUS3 through US11; and RV798 is deleted of US2 through US11.

FIG. 12 provides a summary of MHC class I heavy chain expression datafrom HFF cells infected with wild-type and mutant HCMV. In FIG. 12A, thefirst line is the overall organization of the HCMV wild-type genome, andthe second line is an expansion of the wild-type HindIII-Q and -Xregions of the S component. The ORFs are indicated by an unshadedrectangle; the unlabeled ORF overlapping US4 and US5 is US4.5. Thelocation of the loci which contain gene(s) which are sufficient for MHCclass I heavy chain down-regulation are shown by black rectangles. Onelocus contains ORFs US2-US5 (bases 193119-195607) and the other locus isUS11 (bases 199716-200360). In FIG. 12B, the deletions within thevarious HCMV mutants are indicated by the shaded rectangle. AD169 is thewild-type strain and has no deletions; RV798 is deleted of US2-US11;RV699 is deleted of US11 only; and RV35 is deleted of US6-US11. In FIG.12C, RV8146 is deleted of US4-US11 and RV8173 is deleted of US3-US11.MHC class I heavy chain down-regulation results are fromimmunoprecipitation experiments using the anti-human MHC class I heavychain conformation-independent monoclonal antibody, TP25.99 andmetabolically radiolabeled proteins from HCMV wild-type- ormutant-infected cells.

FIG. 13A-13D show RNA and protein expression from US2. In all theexperiments depicted in FIGS. 13A-D, the multiplicity of infection was5. For FIGS. 13A and 13B, total cytoplasmic RNA was harvested fromuninfected (U) or infected HFF at the indicated hour post-infection (hp.i.), electrophoresed in 1.2% agarose, transferred to nylon membranes,and hybridized with a US2-specific single-stranded riboprobe (i.e.Northern blot analysis). Cells were infected with either HCMV wild-type(AD169) or the US11 -US2 deletion mutant RV798, as indicated. The 72 hp.i. RNA sample from RV798-infected cells (FIG. 13B, lane 7) wasincluded as a negative control and thereby establishes validity to thesmall amount of the 0.9-kb US2 mRNA detected in the 72 h p.i. samplefrom AD169 (FIG. 13B, lane 6). For FIG. 13C, total cellular proteinsfrom uninfected (U) or HCMV wild-type-infected (AD169) HFF cells at theindicated h p.i. were electrophoresed in 15% SDS-PAGE, transferred tonitrocellulose membranes, and probed with anti-US2 polyclonal antisera(i.e., Western blot analysis). The position of the ˜24-kDa US2-encodedprotein (pUS2) is indicated. For FIG. 13D, Western Blot analysis fromHCMV wild-type- and mutant-infected cells was performed as described forFIG. 13C, except that all infected cell proteins were harvested at 11 hp.i.

FIG. 14A-14D show analysis of heavy chain expression in cells infectedwith HCMV mutants at early times post-infection. HFF cells wereuninfected (U) or infected with the indicated virus (multiplicity ofinfection of 5 PFU/cell) and radiolabeled for 4 h from 6-10 hpost-infection. Proteins were harvested immediately after radiolabeling.FIG. 14A is a radiograph of class I heavy chains which wereimmunoprecipitated using TP25.99 murine monoclonal antibody. FIG. 14B isa radiograph in which, to verify approximately equal infection, the72-kDa IE1 immediate-early protein was immunoprecipitated using themurine monoclonal antibody 9221. FIG. 14C is a radiograph of theimmunoprecipitation of the cellular transferrin receptor with murinemonoclonal antibody Ber-T9 to verify approximately equal expression ofthis glycoprotein. FIG. 14D is a radiograph of total radiolabeledproteins to verify approximately equivalent radiolabeling efficiency.

FIG. 15A-15B are Western blots of cell lines expressing the HCMV US11gene. Uninfected human U373-MG cells stably-transfected with a US11expression plasmid were analyzed by Western Blot analysis for MHC classI heavy chain expression (FIG. 15A) and for US11 expression (FIG. 15B)using the TP25.99 monoclonal antibody and the US11 polyclonal antisera,respectively.

FIG. 16A-26B show Western blot analysis of stably-transfected cellproteins. Total protein extracts from either U373-MG parental orstably-transfected (55 series) cells were electrophoresed and blotted asdescribed for FIG. 13C. The blot was cut horizontally such that theportion of the blot containing MHC class I heavy chains (HC) wereanalyzed using the heavy chain monoclonal antibody TP25.99 (FIG. 16A),and the portion of the blot containing the US2-encoded protein (pUS2)were analyzed using the US2 polyclonal antibody (FIG. 16B). 55-212 and55-215 are negative cell lines. Cell line 55-302, although transfectedwith the US2 expression plasmid does not express detectable amounts ofpUS2. Cell lines 55-303, 55-304, and 55-310 express readily detectableamounts of pUS2. The cumulative data indicate the inverse relationshipbetween levels of pUS2 and MHC class I heavy chains.

FIG. 17A-17C show the instability of class I heavy chains in thepresence of pUS2. In FIG. 17A, HFF cells were infected with RV35(deleted of US11 through US6; US2 is retained) at a multiplicity ofinfection of 5. At 3 days p.i., the cells were pulse-labeled for 0.5 hand infected cell proteins were either harvested immediately (P), orharvested after a chase period, either 0.5, 1, 2, or 3 h, in unlabeledmedia. Heavy chains (HC) were immunoprecipitated using the TP25.99monoclonal antibody. The half-life of class I heavy chains inRV35-infected cells is about 0.5 h. Conversely, parallel experimentsusing uninfected or RV798-infected cells (not shown) indicated the heavychains have a half-life of greater than 3 h. In FIG. 17B, similarpulse-chase radiolabeling-immunoprecipitation experiments were performedon U373-MG parental cells or stably-transfected cell lines 55-212 and55-310. Unlike the 55-310 cell line which expresses readily detectableamounts of pUS2, neither U373-MG cells or the 55-212 cell line expressesUS2. Class I heavy chains are stable in cells which do not express US2,but has a short half-life in pUS2-expressing cells (i.e. 55-310). InFIG. 17C, the same pulse-chase extracts used in FIG. 17B were used for acontrol immunoprecipitation by the Ber-T9 monoclonal antibody, which isspecific for another cellular glycoprotein, the transferrin receptor(TfR). In all three cell lines, the stability and processing of thetransferrin receptor is similar.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A recombinant HCMV mutant called RV670 has been constructed whichexpresses a marker gene (β-glucuronidase) in place of a group of viralgenes (Jones and Muzithras, 1992). Upon infection of human fibroblastcells with this mutant, expression of the major histocompatibilitycomplex (MHC) class I heavy chains is not reduced as it is whenwild-type HCMV infects these cells.

Unlike wild-type HCMV, the present invention's recombinant HCMV mutantdoes not result in the down-regulation of cellular MHC class I heavychain protein expression. Surprisingly, it has been found that a 7-kbregion of the HCMV genome contains genes which are required fordown-regulation of heavy chain expression and utilized in the invention.As described more fully below, are two genetic loci of HCMV in this 7-kbregion which are independently sufficient for MHC class I heavy chaindown-regulation. The US11 gene is one such locus, and the other locus isthe US2-US5 gene subregion (i.e., subregion A). Both of these loci arewithin the 7-kb region of the HCMV genome from US2-US11, inclusive,which is deleted from the recombinant virus, RV798 (FIGS. 9 and 12).Correspondingly, RV798-infected cells do not down regulate MHC class Iheavy chains. By the construction and analysis of other defined HCMVmutants, one locus has been found to be defined by the US11 gene, whichwas confirmed by other studies. US11 was expressed constitutively, atvarying levels, in several stably-transfected cell lines. Analysis ofprotein expression in these cell lines indicated that expression of US11was inversely correlated with that of MHC class I heavy chains. It hassubsequently been found that the HCMV gene sufficient for MHC class Idown-regulation within the second locus (US2-US5) is US2.

One skilled in the art will appreciate that efficient antigen processingand presentation is required to activate and expand cytotoxicT-lymphocyte precursors for an efficient cell mediated immune response.Efficient viral antigen presentation requires the continued expressionof MHC class I proteins throughout infection. Infection of cells withRV670 results in continued expression of stable class I heavy chains.

One skilled in the art will appreciate that the virus (RV670) or anotherhuman cytomegalovirus with a deletion of similar genes (e.g. RV798), canbe utilized to produce an effective live vaccine because class I heavychains are still expressed in RV670-infected cells, as they are inuninfected cells, and therefore viral antigen presentation for thepurpose of initiating a cytotoxic T cell response occurs.

In the present invention, flow cytometry and immunofluorescenceexperiments confirmed that cell surface expression of class I heavychains are greatly reduced at late times post-infection in HCMVwild-type strain AD169 infected HFF cells.Radiolabeling-immunoprecipitation experiments indicated thatdown-regulation of newly synthesized MHC class I heavy chains occurredthroughout the course of infection, beginning at very early times (3 h)post-infection (FIG. 4C). This reduction has been reported to be at thepost-translational level: class I heavy chains have a higher turnoverrate in HCMV-infected cells than in uninfected cells (Beersma et al.,1993). Such instability of class I heavy chains results in a reducedcell-mediated immune response to HCMV infection since viral peptideswill be inefficiently presented. Thus, the reduction in class I heavychain expression is important in terms of evasion of a host's immunesystem in the establishment of persistent or latent infections by HCMV(Gooding, 1992).

A bank of HCMV mutants, which represent 18 ORFs which are dispensablefor viral replication in tissue culture, were screened for their abilityto cause down-regulation of MHC class I heavy chains. A 7-kb region ofthe S component of the HCMV genome, containing ORFs US2-US11 (bases193119-200360), was clearly shown to contain genes which are requiredfor this phenotype (data summarized in FIG. 9). Within this region,there are two subregions, each of which contain genes sufficient forheavy chain down-regulation.

Subregion A contains ORFs US2-US5 (bases 193119-195607). It has beenproposed that US2 and US3 encode membrane glycoproteins (Chee et al.,1990). US3 is a differentially spliced gene which is expressedthroughout the viral replicative cycle and encodes a protein withtranscriptional transactivating function (Tenney and Colberg-Poley,1991; Colberg-Poley et al., 1992; Tenney et al., 1993; Weston, 1988).Several smaller ORFs are also present in this subregion (between theORFs US3 and US5), but their expression characteristics or functionshave not been reported. Gretch and Stinski (1990) reported that there isa 1.0-kb early mRNA transcribed from this region of the HCMV genome, butit was not fine-mapped. Now, for the first time, it has been found thatit is expression of the US2 gene which is sufficient for MHC class Idown-regulation within this locus (US2-US5).

Subregion B, which is also sufficient for MHC class I heavy chainreduction, contains the US10 and US11 genes (FIG. 9), bases199083-200360. However, based on data using HCMV mutant RV670 whichexpresses wild-type levels of the US10 gene product, US10 expression isnot sufficient for down-regulation of heavy chain expression (FIG. 5A).The genetic data implicated the US11 gene product as being required. Itis demonstrated herein that US11 expression is sufficient to cause MHCclass I heavy chain down-regulation in stably-transfected uninfectedcells in the absence of other HCMV proteins (FIG. 15). RNA and proteinexpression from US11 begins early and proceeds throughout the course ofinfection (Jones and Muzithras, 1991). US11 encodes a glycoprotein of32-kDa (gpUS11) which has N-linked sugar residues that areendoglycosidase H sensitive. Immunofluorescence experiments show thatgpUS11 is not present on the cell surface, but is detected in thecytoplasm of HCMV-infected cells (FIG. 10). Thus, gpUS11 is retained inthe endoplasmic reticulum or cis golgi. The characteristics of HCMVgpUS11 are similar to the 25-kDa glycoprotein (E3-19K) encoded from theE3 region of adenovirus type 2. Ad E3-19K is nonessential for viralreplication. It has been shown to contain endoglycosidase H-sensitiveN-linked sugar residues, be retained in the endoplasmic reticulum, andbind MHC class I heavy chains, thereby preventing their transport to thecell surface 9 (Anderson et al., 1985; Burgert and Kvist, 1985). Incontrast to Ad E3-19K, a direct association between gpUS11 and class Iheavy chains (i.e., by coimmunoprecipitation) was not detected (data notshown).

The identification of the US2-US11 gene region as the region of the HCMVgenome required for down-regulation of MHC class I heavy chains issignificant in several respects. As mentioned above, expression fromthis region of the genome throughout the course of infection acts tointerfere with an effective cell mediated immune response. Surfaceexpression of MHC class I molecules is required for antigen presentationto activate and expand cytotoxic T lymphocyte (CTL) precursorspopulations (Schwartz, 1985). In addition, they are further required fortarget recognition by the activated CTLs (Zinkernagel and Doherty,1980). In MCMV, CTLs against the major immediate-early protein areprotective against lethal infection by this virus (Jonjic et al., 1988).However, in HCMV-infected individuals, the frequency of CTLs against theanalogous HCMV immediate-early protein, IE1, are reported to beextremely rare (Gilbert et al., 1993). Recent studies have shown that IEpeptides are more efficiently presented by interferon-treatedHCMV-infected cells, than by untreated infected cells (Gilbert et al.,1993). Interferon V causes increased surface expression of MHC class Iproteins. Thus, increasing the expression of class I heavy chains inHCMV-infected cells may be important in the efficient generation ofIE-specific CTLs, or CTLs against other important HCMV antigens. A HCMVmutant deleted of the US2-US11 gene region would have this effect sincethe class I heavy chains are not down-regulated when cells are infectedwith this mutant. Therefore, a deletion of this region of the viralgenome is important in the development of a live HCMV vaccine to inducean effective anti-HCMV immune response.

The elucidation of the US2 and US11 gene products as being sufficientfor class I down-regulation is significant for several reasons based onthe fact that class I proteins mediate the activation of, andrecognition of target cells by, cytotoxic T lymphocytes, the primaryplayer in the cellular immune response. US2 and US11, as genes or,perhaps, as proteins, may be incorporated in clinical treatmentstrategies when expression of cellular MHC class I is undesirable: genetherapy vectors (e.g., adenovirus vectors) and to reduce allograftrejection. US2 and US11 can be used as tools to identify other cellularproteins which may interact with class I heavy chains and thereby effectclass I heavy chain protein stability, processing, and transport to thecell surface. In an HCMV vaccine strategy using a live virus, removal ofUS2, or US11, or both may yield a virus which is a better immunogen thana virus which contains these genes.

Several years ago it was reported that the HCMV UL18 ORF encoded aprotein which resembled MHC class I heavy chains (Beck and Barrell,1988). It was hypothesized that the down-regulation of heavy chains inHCMV-infected cells was due to competition of the UL18 gene product for, β2-microglobulin, which effectively prevented the normal associationof class I heavy chains and β2microglobulin (Browne et al., 1990). Thishypothesis was essentially dispelled when a HCMV mutant deleted of UL18retained its ability to down-regulate heavy chain expression (Browne etal., 1992). It remained possible that the UL18 gene product was only oneof several HCMV genes whose expression is sufficient for this phenotype.However, the present invention data indicates that only genes within theUS2-US11 region are sufficient for class I heavy chain down-regulation.

The existence of two independent mechanisms which result indown-regulation of MHC class I expression emphasizes the importance ofthis phenotype for successful infection and persistence in the host. Onemechanism may serve as a backup system for the other, but it is alsoplausible that there is cell type specificity for each system. Such asituation exists with herpes simplex virus. It was recently reportedthat the 88 amino acid US12 gene product (ICP47) is sufficient for classI heavy chain sequestering in the endoplasmic reticulum (York et al.,1994). However, expression of heavy chains is not affected in herpessimplex virus-infected mouse cells, although ICP47 is expressed in thosecells and murine heavy chains are down-regulated when expressed in anHSV-infected human fibroblast system (York et al., 1994).

A pharmaceutical composition may be prepared containing the recombinantHCMV mutant of the present invention in which the region of the HCMVgenome capable of down-regulating MHC Class I expression in infectedcells has been deleted. The deleted region of the HCMV genome ispreferably open reading frame US2, US11, or both. A stabilizer or otherappropriate vehicle may be utilized in the pharmaceutical composition.

As discussed earlier, the recombinant HCMV mutant of the presentinvention from which a region of the HCMV genome capable ofdown-regulating MHC class I expression has been deleted, may be used ina vaccine for the prevention of cytomegalovirus infections. The deletedregion of the HCMV genome is preferably open reading frame US2, US11, orboth. The vaccine comprises an effective amount of the recombinant HCMVmutant in a pharmaceutically acceptable vehicle. An adjuvant may beoptionally added to the vaccine.

A method of immunizing an individual against cytomegalovirus may becarried out by administering to the individual an immunogenic amount ofthe recombinant HCMV mutant of the present invention which is devoid ofthe gene sequence capable of down-regulating MHC class I expression. Thegene sequence which has been deleted is preferably the region containingopen reading frame US2, US11, or both.

A method of preventing or reducing susceptibility in an individual toacute cytomegalovirus may be carried out by administering to theindividual an immunogenic amount of the recombinant HCMV mutant of thepresent invention which is devoid of the gene sequence capable ofdown-regulating MHC class I expression. The gene sequence which has beendeleted is preferably the region containing open reading frame US2,US11, or both.

Down-regulation of MHC class I expression in a cytomegalovirus infectedcell may be controlled by a method having the steps of identifying agene sequence capable of down-regulating the major histocompatibilitycomplex and deleting the identified gene sequence from thecytomegalovirus genome.

As discussed earlier, the gene sequence involved in the MHC class Iheavy chain down-regulation can be incorporated into adenovirus vectorsor similar virus-based gene therapy vectors to minimize the immuneresponse and allow the use of the vectors in gene therapy. Onevirus-based gene therapy vector comprises the gene sequence of the openreading frame of US2. Another virus-based gene therapy vector comprisesthe gene sequence of the open reading frame of US11. Another virus-basedgene therapy vector comprises the gene sequences of subregions A and B(open reading frames US2-US5 and US10-US11, respectively).

EXAMPLE 1 Virus and Cells

HCMV strain AD169 is obtained from the American Type Culture Collectionand propagated according to standard protocols known by those skilled inthe art. Human foreskin fibroblast (HFF) cells were isolated in thislaboratory and used below passage twenty (Jones and Muzithras, 1991).They were grown in Dulbeccos modified Eagle medium (DMEM) containing 10%fetal bovine serum and 25 mM HEPES.

DNA Sequence

The numbering system of Chee et al. (1990) of the HCMV strain AD169 DNAsequence (Genbank accession number X17403) is used in the presentinvention.

Plasmids

Plasmids used for creation of HCMV mutants were constructed using themethod described previously (Jones et al., 1991; Jones and Muzithras;1992). Generally, the β-glucuronidase reporter gene is surrounded oneach side by 1.5-kb of HCMV sequences which flank the gene(s) to bedeleted from the virus. In each case, the plasmid DNA is linearized witha restriction enzyme which cuts within the prokaryotic backbone prior totransfection. The HCMV strain AD169 genomic DNA fragments are derivedfrom either pHind-G, pHind-X, or pXba-P which contain the HindIII-G(bases 176844 to 195837), -X (bases 195837 to 200856), and XbaI-P (bases200391 to 206314) DNA fragments, respectively (Oram et al., 1982; Joneset al., 1991). pUS7/US3 contained the 1.7-kb PstI-PstI HCMV fragment(bases 196447 to 194741 in pIBI30 vector International Biotechnologies,Inc.!) derived from pHind-G and pHind-X.

Replacement of ORFs IRS1 through US9 and US11 (but not US10) byβ-glucuronidase and plasmid vector sequences (i.e., RV670) was describedpreviously (Jones amd Muzithras, 1992).

To replace HCMV ORFs US11 through IRS1 by β-glucuronidase (i.e., RV7186;FIG. 1B), pBgdUS11/IRS1 was constructed. Sequentially, this plasmidcontained the 1.8-kb fragment PstI-XbaI fragment (bases 202207 to200391, containing US13, US12, and US11 promoter sequences, frompXba-P), β-glucuronidase, a 288-b SV40 fragment containing the early andlate polyadenylation signals (from pRcCMV Invitrogen!), and the 1.7-kbNcoI-NcoI fragment (bases 189763 to 188062, containing J1I to IRL1sequences, from pHind-G).

To replace HCMV ORFs US11 through US2 by, β-glucuronidase (i.e., RV798;FIG. 1C), pBgdUS11/US2 was constructed. Sequentially, this plasmidcontained the 1.8-kb fragment PstI-XbaI fragment (bases 202207 to200391, containing US13, US12, and US11 promoter sequences, frompXba-P), β-glucuronidase, a 255-b fragment containing the US10polyadenylation signal (bases 199276 to 199021, from pHind-X), and the1.3-kb NheI-ApaI fragment (bases 193360 to 192033, containing C-terminalUS2 to IRS1 sequences, from pHind-G).

To replace HCMV ORFs US11 through US6 by, β-glucuronidase (i.e., RV35;FIG. 1D), pBgdUS11/US6 was constructed. Sequentially, this. plasmidcontained the 1.8-kb PstI-XbaI fragment (bases 202207 to 200391,containing US13, US12, and US11 promoter sequences, from pXba-P),β-glucuronidase, and the 1.5-kb HpaI-SstII fragment (bases 195589 to194062, containing C-terminal US6 to US3 sequences, from pHind-G).

Replacement of HCMV ORFs US11-US10, or ORF US11 (singly), byβ-glucuronidase (i.e., RV67 and RV699, respectively) were describedpreviously (Jones et al., 1991). In addition, replacement of HCMV ORFsUS9-US8, US7 (singly), or US6 (singly), by β-glucuronidase (i.e., RV80,RV725, and RV69, respectively) were described previously (Jones andMuzithras, 1992).

To replace HCMV ORFs US9 through IRS1 by , β-glucuronidase (i.e. RV7181;FIG. 1E), pBgdUS9/IRS1 was constructed. Sequentially, this plasmidcontained the 1.1-kb SaII-ApaI fragment (bases 200171 to 199021), the351-b SV40 early promoter (from pRcCMV), β-glucuronidase, the 288-b SV40polyadenylation signal fragment, and the 1.7-kb NcoI-NcoI fragment(bases 189763 to 188062, containing J1I to IRL1 sequences, frompHind-G).

To replace HCMV ORFs US6 through IRS1 by, β-glucuronidase (i.e., RV7177;FIG. 1F), pBgdUS6/IRS1 was constructed. Sequentially, this plasmidcontained the 1.7-kb NcoI-NcoI fragment (bases 188062 to 189763,containing IRL1, J1I, and IRS1 promoter sequences, from pHind-G),β-glucuronidase, the 255-b fragment containing the US10 polyadenylationsignal (bases 199276 to 199021, from pHind-X), and the 1.8-kb BsmI-SauIfragment (bases 196222 to 198030, containing US7 to C-terminal US9sequences, from pHind-X).

To replace HCMV ORFs US3 and US2 by β-glucuronidase (i.e., RV47; FIG.1G), pBgdUS3/US2 was constructed. Sequentially, this plasmid containedthe 1.7-kb PstI-PstI fragment (bases 196447 to 194741), a 180-bSmaI-HaeIII fragment containing the HSV-1 gH promoter (McKnight, 1980),β-glucuronidase, the 255-b US10 polyadenylation signal fragment, and the1.3-kb NheI-ApaI fragment (bases 193360 to 192033, containing C-terminalUS2 to IRS1 sequences, from pHind-G).

To replace HCMV ORF US1 by, β-glucuronidase (i.e., RV5122; FIG. 1H),pBgdUS1 was constructed. Sequentially, this plasmid contained the 1.8-kbAatII-SstI fragment (bases 190884 to 192648, containing IRS1 and US1C-terminal sequences, from pHind-G), a 180-b SmaI-HaeIII fragmentcontaining the HSV-1 gH promoter (McKnight, 1980), β-glucuronidase, the255-b US10 polyadenylation signal fragment, and the 1.6-kb SphI-SphIfragment (bases 192934 to 194544, containing US2 and C-terminal US3sequences, from pHind-G).

To replace HCMV ORF IRS1 by β-glucuronidase (i.e., RV46; FIG. 11),pBgdIRS1 was constructed. Sequentially, this plasmid contained the1.7-kb NcoI-NcoI fragment (bases 188062 to 189763, containing IRL1, J1I,and IRS1 promoter sequences, from pHind-G), β-glucuronidase, the 255-bfragment containing the US10 polyadenylation signal (bases 199276 to199021, from pHind-X), and the 1.2-kb NarI-XhoI fragment (bases 191830to 193003, containing C-terminal IRS1 and US1 sequences, from pHind-G).

To delete HCMV ORFs US11 through US2 without insertion of a reportergene (i.e., RV799; FIG. 1J), pdUS11/US2 was constructed. Sequentially,this plasmid contained the 1.8-kb fragment PstI-XbaI fragment (bases202207 to 200391, containing US13, US12, and US11 promoter sequences,from pXba-P), β-glucuronidase, 65-b NruI-ApaI fragment containing theUS10 polyadenylation signal (bases 199086 to 199021, from pHind-X), andthe 1.3-kb NheI-ApaI fragment (bases 193360 to 192033, containingC-terminal US2 to IRS1 sequences, from pHind-G).

To replace HCMV ORFs US11 through US4 by, β-glucuronidase (i.e., RV8146;FIG. 2B), pBgAUS11/US4 was constructed. Sequentially, this plasmidcontains the 1.8-kb fragment PstI-XbaI fragment (bases 202207 to 200391;containing US13, US12, and US11 promoter sequences; from pXba-P),β-glucuronidase, the 180-b SmaI-HaeIII fragment containing the HSV-1thymidine kinase polyadenylation signal (McKnight, 1980), and the1.662-kb EcoRV-SmaI (bases 195083 to 193421; containing US3 and US2sequences; from pHind-G).

To replace HCMV ORFs US11 through US3 by , β-glucuronidase (i.e.,RV8173; FIG. 2C), pBgΔUS11/US3 was constructed. Sequentially, thisplasmid contains the 1.8-kb fragment PstI-XbaI fragment (bases 202207 to200391; containing US13, US12, and US11 promoter sequences; frompXba-P), β-glucuronidase, the 180-b SmaI-HaeIII fragment containing theHSV-1 thymidine kinase polyadenylation signal (McKnight, 1980), and the1.464-kb KpnI-SacI (bases 194112 to 192648; containing US2 and US1sequences; from pHind-G).

Isolation of Recombinant Mutant HCMV

Creation and isolation of recombinant mutant HCMV was done as describedpreviously (Jones et al., 1991; Jones and Muzithras, 1992). HFF cellswere split so that they were 70-80% confluent on the day oftransfection. The cells were trypsinized and suspended to 5.6×10⁵ cellsper ml in DMEM/10% FCS/25 mM HEPES. The DNA was transfected using amodified calcium phosphate co-precipitation technique. 1.5 μg ofinfectious HCMV DNA and 2.5 μg of linearized plasmid DNA were mixed inthe calcium chloride solution (300 μl containing 10 mM Tris pH 7.0/250mM calcium chloride) and chilled on ice. To initiate theco-precipitation, the DNA was removed from the ice and 300 μl 2× HeBS pH6.95 (at room temperature; 1× HeBS was 19.2 mM HEPES, 137 mM NaCI, 5 mMKCI, 0.8 mM sodium phosphate, 0.1% dextrose) was added dropwise withgentle mixing. After 1.5 minutes, the precipitate was placed on ice (toprevent further precipitate from forming). The precipitate was mixedwith 3×10⁶ cells (in suspension) and placed in a 82 mm tissue cultureplate. After 6 h at 37° C., the media was removed and the cells wereshocked with 20% DMSO in 1× HeBS for 2 minutes. The cells were washedtwice with PBS and growth media was added. The media was changed every4-7 days. After 14 days, viral plaques were observed and the cells wereoverlaid with 0.5% agarose in DMEM containing 150 μg/ml X-gluc (5-bromo4-chloro 3-indol 1-glucuronide; Biosynth). Blue plaques (i.e.,β-glucuronidase-positive mutant virus plaques) were picked several daysafter adding the overlay. Recombinant viruses were plaque purified threetimes. HCMV mutant RV799 was, β-glucuronidase-negative and was isolatedusing a modification of the above procedure. In this case,β-glucuronidase-positive HCMV mutant RV134 was the parent virus (Joneset al., 1991). Thus, RV134 genomic DNA was used instead of wild-typestrain AD169 DNA in the transfections. Primary plaques appearing on theprimary transfection plates were picked at random and replated on HFFcells. After 10 days, the media was removed and the infected cells wereoverlaid with X-gluc-containing agarose as described above. In thiscase, white plaques (β-glucuronidase-negative mutant virus plaques) werepicked 4 days later and plaque purified. The proper genomic organizationof each of the HCMV mutants was verified by DNA blot hybridizationanalysis as described previously (Jones et al., 1991).

Antibodies

Rabbit polyclonal antisera reactive with HCMV US11 proteins and HCMVUL80 proteins are described previously (Jones et al., 1991; 1994).Murine monoclonal antibodies W6/32, specific for aconformation-dependent epitope on the heavy chain of human MHC class Iproteins, and Ber-T9, specific for the human transferrin receptor, werepurchased. Murine monoclonal antibody TP25.99 (D'Urso et al., 1991),specific for a conformation-independent epitope on the heavy chain ofhuman MHC class I proteins, was obtained from Dr. S. Ferrone (Departmentof Microbiology, New York Medical College, Valhalla, N.Y.). Murinemonoclonal antibody 9221, specific for the HCMV IE1 protein, waspurchased from Dupont.

US2 polyclonal antisera was obtained by isolating a US2-glutathioneS-transferase fusion (GST) protein which was subsequently used as theimmunogen in rabbits. Specifically, the portion of the HCMV US2 geneencoding amino acids 20 through 110 was generated by polymerase chainreaction as a NcoI/EcoRI fragment and fused in frame with the C-terminusof the GST protein in the pGST(Nco) vector to yield the plasmidpGST(Nco)-US2. The vector pGST(Nco) was modified from the glutathioneS-transferase fusion vector pGEX-2T (Pharmacia stock no. 27-4801-01) bydigestion with SmaI and the addition of NcoI linkers such that the openreading frame was retained. The plasmid PGST(Nco)-US2 was introducedinto E. coli strain DH5 and fusion protein synthesis was induced withIPTG. The GST-US2 fusion protein was isolated from sonicated E. coli bybinding to and elution from glutathione sepharose 4B (Pharmacia) asdescribed by the manufacturer. One hundred microgram aliquots of theGST-US2 fusion protein were used as an immunogen in female New Zealandwhite rabbits to generate US2 polyclonal antisera.

Radiolabeling and Immunoprecipitation of Infected Cell Proteins

Pulse-chase radiolabeling was done according to standard protocol(Sambrook et al., 1989). HCMV-infected HFF cells (multiplicity ofinfection equalled five) was pulse-labeled with 200 μCi of ³⁵ S !methionine and ³⁵ S ! cysteine (NEN-DuPont) per ml inmethionine/cysteine-free Dulbecco's modified Eagle medium (DMEM) at theindicated time period post-infection. The radioactive media was removed,the cells washed twice in complete DMEM, and chases were done for theindicated time in complete DMEM. Proteins were extracted using tripledetergent lysis buffer (Sambrook et al., 1989). The cleared proteinextracts (supernatant after centrifugation for 5 minutes at 15000×g and4° C.) were retained for immunoprecipitation according to standardprotocol (Sambrook et al., 1989). Proteins binding to antibodies werepelleted using protein A sepharose (Pharmacia). For immunoprecipitationsof the human transferrin receptor, rabbit anti-mouse IgG (Pierce) wereadded prior to protein A sepharose. The washed immunoprecipitates wereboiled in the presence of 2-mercaptoethanol and electrophoresed indenaturing polyacrylamide gels. The gels were fixed and soaked in 1Msodium salicylate fluor (Sambrook et al.,1989) prior to drying andautoradiography.

Immunofluorescence

Immunofluorescence assays were done according to standard protocol(Harlow, 1989). All procedures were done in 60 mm tissue culture plates.Briefly, infected or uninfected HFF cells were fixed with 4%paraformaldehyde and permeabilized with 0.2% Triton X-100 (whereindicated). After adding 3% bovine serum albumin in phosphate-bufferedsaline, the cells were held overnight at 4° C. The cells were treatedsequentially with the following antisera, each for 30 minutes at roomtemperature: 10% HCMV-negative human serum (to block any Fc receptors);the indicated primary antibody; and FITC-conjugated anti-mouse oranti-rabbit IgG, as appropriate.

EXAMPLE 2 Class I Down-Regulation in HCMV-lnfected Human Fibroblasts

The timing and nature of MHC class I heavy chain down-regulation wasascertained in the human foreskin fibroblast (HFF) cell culture system.By flow cytometry, HCMV strain AD169 wild-type-infected HFF cells weresignificantly reduced in the expression of class I heavy chains on theircell surface at late times post-infection (i.e., 72 h) using theconformation-dependent class I monoclonal antibody W6/32 (FIG. 3). InWestern analyses using the conformation-independent class I monoclonalantibody (TP25.99), it was demonstrated that the steady state level ofclass I protein was also reduced at late times post-infection (FIG. 4A).Because viral peptides are presented at the cell surface by-class Icomplexes assembled after infection, the status of class I proteinssynthesized at various times post-infection was assessed byimmunoprecipitation of metabolically radiolabeled proteins. As shown inFIG. 4B, reduction in expression of class I heavy chains was detectedboth in the presence and absence of the viral DNA synthesis inhibitor,phosphonoformate. This indicated that viral immediate-early or earlygene functions are sufficient for heavy chain reduction. In addition, itwas demonstrated that heavy chain down-regulation was detected at veryearly times post-infection: 3 h (FIG. 4C). Since this effect wasobserved using the conformation-independent antibody, the reductionreflects overall levels of newly synthesized heavy chains.

Screening of HCMV Mutants for the Loss of MHC Class I Down-Regulation

Several previously constructed HCMV deletion mutants, representing 18nonessential ORFs (UL33, UL81, IRS1, US1-US13, US27-US28, and TRS1),were screened for heavy chain expression by flow cytometry andimmunoprecipitation analyses. Only RV670, a mutant deleted of a 9-kbregion within the S component of the HCMV genome (Jones and Muzithras,1992), did not retain the wild-type down-regulation phenotype (FIG. 5A).This mutant was deleted of at least 11 ORFs, IRS1 through US11 (exceptfor US10), which includes the US6 family of genes (US6-US11 ) whichputatively encode glycoproteins (Chee et al., 1990). To confirm thisobservation, two additional independently derived mutants which had thesame deletion as RV670 and a new mutant, RV7186, deleted of the entireIRS1-US11 region (FIG. 1) were tested. Each was phenotypically identicalto RV670 and stably expressed class I heavy chains. Previously, weconstructed HCMV mutants deleted of US6 family ORFs, either individuallyor in groups (Jones and Muzithras, 1992), and similar deletion mutantswithin the adjacent IRS1-US3 region. By immunoprecipitation using theconformation-independent antibody, all of these mutants were shown toretain the ability to down-regulate class I heavy chains (FIG. 5A) atlate times post-infection in HFF cells. Control experiments indicatedthat radiolabeling was equivalent between the different infected cellcultures (FIG. 5B) and that infection proceeded to late times equally,as judged by pp65 (FIG. 5B) and UL80 protein (FIG. 5C) expression. Thesedata indicated: (i) that more than one viral gene is sufficient for thereduction in class I heavy chains; or (ii) gene(s) between US3 and US6,deleted in RV670 and RV7186 but not the other mutants, is required forthe phenotype.

Identification of a 7-kb Region of the HCMV Genome Required for MHCClass I Down-Regulation

To further localize the region containing gene(s) involved in MHC classI heavy chain down-regulation, additional HCMV replacement mutantscontaining deletions of multiple genes within the IRS1-US11 gene regionwere created (FIG. 1). One of these mutants, RV798, was deleted of genesfrom US2-US11. In HFF cells infected by RV798 and analyzed at late timespost-infection, MHC class I heavy chains were not down-regulated as theyare in wild-type strain AD169-infected cells (FIG. 5A); in fact, aslight stimulation is observed. Several independently-derived deletionmutants identical to RV798 were examined similarly: all lacked theability to down-regulate class I heavy chains.

To further confirm that the 7-kb HCMV US2-US11 region contained thegene(s) required for heavy chain down-regulation, mutant RV799 wasconstructed which had the identical US2-US11 deletion as RV798, but wascreated by a different strategy. RV798 was derived from wild-type strainAD169 by inserting a, β-glucuronidase marker gene in the place ofUS2-US11. In contrast, the parent of RV799 was RV134, a mutant which was, β-glucuronidase-positive since it had a β-glucuronidase expressioncassette inserted within the US9-US10 intergenic region (Jones et al.,1991). To create RV799, a plasmid was designed which upon recombinationwith the RV134 genome would simultaneously delete US2-US11 and theβ-glucuronidase expression cassette (FIG. 1J). The proper RV799 HCMVmutant was isolated as a white plaque in the presence of theβ-glucuronidase substrate, since it was β-glucuronidase-negative. RV799,but not the RV134 parent, was phenotypically identical to RV798 (FIG.6). Thus, since RV798 and RV799 were created by different strategiesfrom parents which retained the ability to down-regulate MHC class Iheavy chains, this confirms that the gene(s) required for the phenotypeare located within the 7-kb US2-US11 region (bases 193119-200360).

To determine whether the proper surface expression of class I heavychains occurred at late times post-infection with either RV798 or RV799,immunofluorescence assays were done. Using either theconformation-dependent (W6/32) or conformation-independent (TP25.99)monoclonal antibodies, surface expression of MHC class I heavy chainswas detected in uninfected and RV798and RV799-infected HFF cells, butnot wild-type AD169-infected HFF cells. Proper maturation of class Iheavy chains in uninfected cells yielded endoglycosidase H resistantmolecules. In contrast, class I heavy chains synthesized inAD169-infected cells were reported to be entirely endoglycosidase Hsensitive (Beersma et al., 1993). As shown in FIG. 7, class I heavychains synthesized in RV798infected HFF cells, either at early or latetimes post-infection, were converted to the mature endoglycosidaseH-resistant form at a rate similar to those synthesized in uninfectedcells. Taken together, these data indicate that MHC class I synthesis,processing, and surface expression are not impaired in cells infectedwith these HCMV mutants. Furthermore, the results indicate that the 7-kbregion containing US2-US11 genes contain one or more genes required forheavy chain down-regulation by HCMV.

Two Subregions Within the US2-US11 Gene Region Contain Genes Which areInvolved in Class I Heavy Chain Down-Regulation

The region of the HCMV genome deleted in RV35 was from US6-US11, andUS2-US11 in RV798 (FIG. 1). In RV35-infected HFF cells, MHC class Iheavy chains were down-regulated, but in RV798-infected cells they werenot (FIG. 5A). This data indicates that one or more genes involved inheavy chain down-regulation maps within the 2-kb subregion from ORF US2through US5 (subregion A; bases 193119-195607). To determine if this2-kb subregion is required for class I heavy chain down-regulation, HCMVreplacement mutants RV7181 and RV7177 were examined. HCMV ORFs IRS1-US9and IRS1-US6 are deleted, respectively, in these mutants (FIG. 1);hence, subregion A is absent from both mutants. Experiments in infectedHFF cells at late times post-infection indicated that both mutantsretained the ability to efficiently down-regulate class I heavy geneexpression (FIG. 8). Therefore, when present in the HCMV genome, gene(s)within subregion A are sufficient for reduction of MHC expression (e.g.,RV35), although their presence is not required for the phenotype.Furthermore, the cumulative data (summarized in FIG. 9) indicate thatthere are no HCMV genes within the identified 7-kb US2-US11 region(i.e., the region deleted in RV798) which are absolutely required forefficient heavy chain down-regulation in infected HFF cells, suggestingthat gene(s) from another portion of the US2-US11 gene region are alsosufficient for the phenotype at late times post-infection.

Evidence Indicating That the US11 Gene Product is Involved in MHC ClassI Heavy Chain Down-Regulation

In HFF cells infected with mutant RV7181, deleted from IRS1-US9 (FIG.1), MHC class I heavy chain expression was down-regulated, in contrastto RV798-infected HFF cells (FIG. 8A). This data suggests that a secondsubregion (subregion B), comprised of the US10 and US11 genes (bases199083-200360), is involved in reduction of heavy chain expression.However, the expression of US10 from the context of the HCMV genome isnot sufficient for heavy chain down-regulation. HCMV mutant RV670expressed US10 at steady-state levels similar to wild-type and wasdeleted of all of the other ORFs in the 7-kb US2-US11 gene region, butit did not cause down-regulation of MHC class I heavy chains in infectedHFF cells (FIG. 5A). Thus, US11 is the gene in subregion B which isimplicated by this genetic data.

US11 encodes a 32-kDa glycoprotein (gpUS11) containing N-linked, but notO-linked, carbohydrates which are completely sensitive toendoglycosidase H, indicating that the sugars are in the high mannoseform. gpUS11 was detected throughout infection, beginning at very earlytimes (i.e. 3 h) and continuing through late times post-infection.However, levels of gpUS11 in the infected cell are most abundant atapproximately 8 h post-infection. To determine its location in theinfected cell, rabbit polyclonal antisera (Jones and Muzithras, 1991)was used in immunofluorescence assays of wild-type strain AD169-infectedcells. Uninfected and RV699-infected HFF cells were used as negativecontrols. RV699 is an HCMV mutant which is isogeneic with AD169, exceptfor a deletion of the US11 ORF (Jones et al., 1991). In cells fixed andpermeabilized at 8 h post-infection, cytoplasmic fluorescence whichobscured definition of the nucleus was observed in AD169-infected HFFcells, but not in either negative control cells (FIGS. 10A and 10C,respectively). In general, the specific fluorescence was more intense inthe perinuclear area. There was no specific fluorescence detected innon-permeabilized cells (FIGS. 10B and 10D). The fluorescence andendoglycosidase-H sensitivity data indicate that gpUS11 is not a cellsurface glycoprotein. From the translated DNA sequence, gpUS11 ispredicted to have hydrophobic domains near its N- and C-termini (Westonand Barrell, 1986) which are putative signal sequence and transmembranedomains, respectively. Thus, gpUS11 is associated with intracytoplasmicmembranes, possibly the endoplasmic reticulum or cis golgi.

Evidence Indicating That the US2 Gene Product is Involved in MHC Class IHeavy Chain Down-Regulation

Class I heavy chains were down-regulated in RV35-infected cells, but notin RV798-infected cells (FIGS. 11 and 12). To define the HCMV genewithin subregion A (i.e., US2-US5) which is sufficient for MHC class Iheavy chain down-regulation, several additional HCMV deletion mutantswere constructed and analyzed. These new mutants included RV8146 andRV8173, which were deleted of the gene regions US4-US11 and US3-US11,respectively (FIG. 2). The new mutants were constructed using thepreviously described homologous recombination technique. Class I heavychain expression in cells infected by these viruses was assayed inmetabolic radiolabeling-immunoprecipitation experiments. Unlike RV798infected cells, MHC class I heavy chains were down-regulated in cellsinfected with RV8146 and RV8173 (FIG. 11). The cumulative data derivedfrom these experiments indicated that genes encompassing US3-US5 are notrequired for class I heavy chain down-regulation. Since the genotypicdifference between mutants RV798 and RV8173 is the presence of US2 inthe latter, US2 was therefore implicated in these genetic experiments.

RNA blot analyses indicated that the US2 gene is transcribed throughoutmost of the HCMV replicative cycle, from very early times (e.g., 3 h) tolate times (e.g., 72 h) post-infection (FIGS. 13A and 13B). To analyzeUS2 protein (pUS2) expression directly, a GST-US2 fusion protein wasmade in bacteria and used as an immunogen in rabbits for the generationof polyclonal antisera reactive with pUS2. This antisera reacted with anapproximately 24-Kda protein which was expressed throughout thereplicative cycle in cells infected with HCMV wild-type strain AD169(FIG. 13C). The apparent mass of the pUS2 in these experimentscorrelated well with the 23.1-Kda computer-calculated molecular mass ofthe US2 protein derived from the translated US2 DNA sequence (Chee etal., 1990). The results from the genetic experiments described abovepredicted that pUS2 is expressed by cells infected with deletion mutantsRV8146 and RV8173, but not by RV798. Western Blot analysis of infectedcell proteins confirmed this prediction (FIG. 13D).

Down-Regulation of MHC Class I Expression at Early Times Post-infectionby HCMV Mutants

Down-regulation of MHC class I expression in wild-type strainAD169-infected cells are shown to begin at very early timespost-infection (FIG. 4C). To determine if any of the mutants aredeficient for this early down-regulation, immunoprecipitationexperiments were performed using extracts from infected HFF cellsradiolabeled from 6-10 h post-infection. The level of class I heavychains were reduced during this early period post-infection in HFF cellswith each of the mutants, except for RV798, the mutant deleted of theentire 7-kb US2-US11 region (FIG. 14A). Control experiments demonstratedthat the different mutant-infected cells were equally infected andradiolabeled (FIG. 14B and D). Expression of another cellularglycoprotein, the transferrin receptor, was not differentially affectedby the various mutants (FIG. 14C). Thus, genes required for heavy chaindown-regulation at early times post-infection are the same as thosenecessary for reduction at late times post-infection. Moreover,expression of gene(s) from either subregion identified to be involved indown-regulation of heavy chain expression at late times post-infectionare sufficient for reduction at very early times postin-fection.

EXAMPLE 3 Recombinant HCMV (RV798) Vaccine Preparation

HCMV vaccines were prepared using a method described previously (Elekand Stern, 1974). HCMV mutant RV798 was grown on MRC-5 human diploidlung fibroblasts (CCL171 American Type Culture Collection!) or humanforeskin fibroblasts (MRHF BioWhittaker!). Cells were infected at amultiplicity of infection equal to one in Dulbecco's modified Eaglemedium (DMEM) containing 5% calf serum and 5% fetal calf serum. After 24h, the medium was removed and the cells washed three times with eitherHank's balanced salt solution or Dulbecco's phosphate-buffered saline.Fresh DMEM medium without serum was added; the infected cells wereincubated 4 days after the appearance of late viral cytopathic effect(usually 7 days post-infection). After a preclearing centrifugation step(6,000×g for 20 minutes at 18° C.), cell-free virus was pelleted bycentrifugation at 15,500×g for one hour at 18° C. The pelleted virus wasresuspended in Dulbecco's phosphate-buffered saline containing 25%sorbitol and stored in aliquots at -70° C. The titer of RV798 vaccinestock is determined using standard procedures on human foreskinfibroblasts (Wentwork and French, 1970). The vaccine is administered bysubcutaneous inoculation of approximately 10³ -10⁷ plaque forming unitsinto the deltoid region of the upper arm, as described previously (Elekand Stern, 1974; Gehrz et al., 1980; Starr et al., 1981).

EXAMPLE 4 gpUS11 is Sufficient for Down-Regulation of MHC Class I HeavyChains

The genetic data from the deletion virus-infected cells in Example 2indicated that US11 is the gene within subregion B (i.e., US10-US11) ofHCMV involved in MHC class I heavy chain down-regulation. To determineif the US11 gene product, in the absence of any other viral geneproducts, is capable of causing heavy chain down-regulation, the US11coding region (bases 200360-199716 Chee et al., 1990!) and somenon-coding flanking sequences, encompassing bases 200391-199683, werecloned into a eukaryotic expression plasmid under the transcriptionalcontrol of the constitutive HCMV major immediate-earlyenhancer-promoter. Human U373-MG astrocytoma cells (HTB 17 American TypeCulture Collection!) were transfected with this plasmid (Sambrook et al,1989) and stably-transfected cells were selected in the presence of0.375 μg/ml of puromycin, since the plasmid also encodes for theprokaryotic puromycin resistance gene. Clones were picked and expandedinto cell lines. Those expressing gpUS11 were identified by Western Blotanalysis; different cell lines expressed varying amounts of US11. MHCclass I heavy chain expression in these cell lines was analyzed in asimilar fashion. As shown in FIG. 15, expression of US11 was inverselycorrelated with the expression of class I heavy chains. These data provethat expression of HCMV US11 is sufficient for the down-regulation ofMHC class I heavy chain expression in the absence of any other viral.gene products.

EXAMPLE 5 pUS2 is Sufficient for Down-Regulation of MHC Class I HeavyChains

The genetic data from the deletion virus-infected cells in Example 2indicated that US2 is the gene within subregion A (i.e., US2-US5) ofHCMV involved in MHC class I heavy chain down-regulation. To confirmthis indication, stably-transfected cells which constitutively expresspUS2 were created. In this case, the HCMV US2 coding region (bases193715 to 193119), and some non-coding flanking sequences, encompassingbases 193779 to 193003, were cloned into an expression vector plasmiddesignated pIEsp-puro to yield pIEspUS2-puro. In this plasmid, US2 wasunder the transcriptional control of the constitutive HCMV majorimmediate-early enhancer-promoter. This plasmid also contained apuromycin resistance gene expression cassette. After transfection of theplasmid into U373-MG astrocytoma cells, stably-transfected cell lineswere selected and cloned in the presence of the drug puromycin (0.375μg/ml). As a negative control, a similar plasmid (pIEspBgIuc-puro),which contains the prokaryotic , β-glucuronidase gene instead of US2,was transfected and cell lines were selected and cloned. Steady-stateprotein expression in these cell lines was analyzed by Western blotanalysis.

Similar to the US11-expressing cell lines of Example 4, the expressionof US2 was found to be inversely correlated with expression of thecellular MHC class I heavy chains. Specifically, parental U373-MG cellsand cell lines 55-212 and 55-215 derived from the transfection of thenegative control plasmid did not express US2 and had high levels ofclass I heavy chains (FIG. 16, lanes 1-3). Cell lines 55-303, 55-304,and 55-310 were transfected with pIEspUS2-puro and expressed US2, buthad relatively low levels of class I heavy chains (FIG. 16, lanes 5-7).Another cell line derived from the transfection of pIEspUS2-puro,55-302, did not express US2 (presumably due to the disruption of the US2gene during the generation of this cell line), but expressed high levelsof class I heavy chain (FIG. 16, lane 4). The cumulative datademonstrated that US2 expression causes down-regulation of MHC class Iheavy chain expression.

Data from HCMV-infected cells indicated that MHC class I heavy chainsare down-regulated by a post-transcriptional mechanism resulting in theincreased turnover (i.e., shorter half-life) of these proteins (Beersmaet al., 1994; Yamashita et al., 1994). In cells infected with mutantRV35 (deleted of US6-US11 and containing the US2-US5 locus), thehalf-life of class I heavy chains is about 0.5 h, while the half-life inuninfected and RV798-infected cells is >3 h (FIG. 17A). Since US2 is thegene involved in class I heavy chain down-regulation within the US2-US5locus, class I heavy chains in the stably-transfected US2expressing celllines were expected to have a high turnover rate compared to parentalU373-MG cells and negative control cell lines. To test this prediction,representative cell lines of each type were "pulse" metabolicallyradiolabeled for 0.5 h with ³⁵ S-methioninelcysteine-containing media,and then either harvested immediately or "chased" in unlabeled media forvarious times prior to harvesting. Immunoprecipitation experiments usingthe pulse-chase extracts indicated that class I heavy chains were stable(half-life>3.5 h) in parental U373-MG cells and the negative controlcell line 55-212 (FIG. 17B, lanes 1-4 and 5-8, respectively). Incontrast, class I heavy chains had a very short half-life in theUS2-expressing cell line 55-310 (FIG. 17B, lanes 9-12), as evidenced bythe detection of heavy chains only in the pulse sample.

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What is claimed is:
 1. A cell transformed or transfected with a vectorselected from the group consisting of an adenovirus, adeno-associatedvirus, retrovirus, and herpes simplex virus, said vector comprising agene sequence encoding the open reading frame US2 of the humancytomegalovirus genome, wherein said gene sequence is expressed in saidcell.